1. Field of the Invention
The invention relates to novel vaccines for protecting fish against infection with Streptococcus agalactiae, and a novel process for making the same.
Streptococcus agalactiae is a Group B streptococcal bacterium that causes severe economic losses in a number of species of cultured and wild fish. This infectious bacterium is common in aquaculture facilities where fish are intensively cultured in fresh, brackish, or marine waters. The high densities of fish and the aqueous environment favor the rapid transmission of streptococcal disease. Moreover, infected cultured fish may transmit the disease to wild fish populations, or infected wild fish may transmit the disease to cultured fish.
2. Description of the Prior Art
Vaccines have previously been developed against various Streptococcus and Enterococcus species utilizing strategies based on either intraperitoneal or intramuscular injection. Several injectable vaccines have been developed for the prevention of streptococcosis, although many of these vaccines differ in their formulation. Protection of rainbow trout after intraperitoneal (IP) vaccination with a formalin killed Streptococcus iniae vaccine was reported by Eldar et al. (Development and efficacy of a vaccine against Streptococcus iniae infection in farmed rainbow trout, Vet Immunol Immunopathol 1997; 56: 175–183). Klesius et al. [Efficacy of a killed Streptococcus iniae vaccine in tilapia (Oreochromis niloticus), Bull Eur Ass Fish Pathol 1999; 19(1): 38–41; and Efficacy of a single and combined Streptococcus iniae isolate vaccine administered by intraperitoneal and intramuscular routes in tilapia (Oreochromis niloticus), Aquaculture 2000; 188(3–4): 237–246] have developed a modified killed S. iniae vaccine composed of whole cells and concentrated, extracellular products. Immunized 25 g tilapia (Oreochromis niloticus) had a relative percent survival (RPS) of 95.3 and 100 g tilapia had RPS values ranging from 84.2 to 94. Turbot (Scophthalmus maximus) were protected against Enterococcus sp. after vaccination with a toxoid-enriched bacterin [Romalde et al., Prevention of streptococcosis in turbot by intraperitoneal vaccination: A review, J Appl Ichthyol 1999; 15: 153–158; Long-lasting protection against turbot streptococcosis obtained with a toxoid-enriched bacterin, Bull Eur Ass Fish Pathol, 1996; 16(5): 169–171; and Toranzo et al., Efficacy of intraperitoneal and immersion vaccination against Enterococcus sp. infection in turbot, Aquaculture 1995; 134: 17–27]. The toxoid-enriched bacterin vaccine was a combination of two formalin-killed Enterococcus sp. isolates and their culture fluids. Rainbow trout (Oncorhynchus mykiss) immunized with formalin-killed Streptococcus sp. in Freund's incomplete adjuvant were protected against Streptococcus sp., whereas trout immunized by bath immersion were not protected [Akhlaghi et al., Comparison of passive and active immunization of fish against streptococcosis (enterococcosis), J Fish Dis 1996; 19: 251–258]. Recently, Nakanishi et al. (Development of a new vaccine delivery method for fish: Percutaneous administration by immersion with application of a multiple puncture instrument, Vaccine 2002; 20: 3764–3769) demonstrated the protection of skin punctured juvenile rainbow trout immersed in a formalin killed S. iniae vaccine suspension rivaled that obtained by IP injection.
Eldar et al. disclosed the preparation of an injectable vaccine prepared from formalin-killed Streptococcus difficile. This vaccine was reported to protect tilapia (Oreochromis sp.) against challenge with S. difficile [Eldar et al., Vaccination with whole-cell vaccine and bacterial protein extracts protects tilapia against Streptococcus difficile meningoencephalitis, Vaccine 1995; 13(9): 867–870; and Bercovier et al., Immunization with Bacterial Antigens: Infections with Streptococci and Related Organisms; Fish Vaccinology, Dev. Biol. Stand., Vol. 90 (Liiehaug, G., Midlyng, P J & Brown, F. eds.) Karger, Basel, Switzerland, pp. 153–160, 1997].
In a subsequent report however, Vandamme et al. (Streptococcus difficile is a nonhemolytic group B, type Ib Streptococcus, Int J Syst Bacteriol, 1997; 47(1): 81–85), proposed that the S. difficile reported by Eldar et al. was actually a non-hemolytic, group B Streptococcus, S. agalactiae. Indeed, many of the reported streptococcal fish isolates originally unspeciated or misidentified have been more recently characterized as non-hemolytic, group B Streptococcus, S. agalactiae. 